Research in the Immunopathology Section focuses on the biological mediators and signal transduction pathways involved in the modulation of human monocyte functions that may contribute to the immunopathology associated with various inflammatory lesions. Connective tissue destruction is associated with many diseases in which the monocyte/macrophage is a prominent cell. Examples of these chronic inflammatory lesions include periodontal disease and rheumatoid arthritis where destruction of connective tissue leads to loss of structural integrity and atherosclerosis where degradation of connective tissue in vulnerable plaques leads to rupture and subsequent ischemic events. Since matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are believed to play a major role in the destruction and remodeling of connective tissue, a major emphasis has been placed on how these enzymes and inhibitors are regulated in the human monocyte. Biological mediators involved in the regulation of monocyte matix metalloproteinases: Implication for chronic inflammatory diseases Our studies on biological mediators have examined factors associated with cardiovascular disease that may also impact on periodontal disease and rheumatoid arthritis through their effect on monocyte MMP production. Chlamydia pneumoniae has been reported to possibly contribute to the pathological events associated with atherosclerosis. Our findings published in FY 05 demonstrate that C. pneumoniae significantly enhances MMP-1 and MMP-9 by cytokine (tumor necrosis factor-alpha and granulocyte macrophage-colony stimulated factor) activated monocytes through a prostaglandin E2 (PGE2) dependent mechanism. Moreover, exposure to live or killed C. pneumoniae enhance MMP production by activated monocytes. These results indicate that components of C. pneumoniae remaining in tissues at infection sites after clearance of live organisms can continue to enhance activation of monocytes. Moreover, monocytes primed by exposure to C. pneumoniae components may travel to various inflammatory sites where they enhance connective tissue destruction. In a second study we have shown that angiotensin II (Ang II), which mediates hypertension increasing the risk for acute coronary syndrome, is released by cytokine-activated monocytes and has an integral role in the signal transduction pathway that leads to the production of MMP-1 by monocytes. Ang II mediated increase in MMP-1 synthesis by monocytes occurred only in conjunction with cytokine stimulation. Ang II was shown to mediate its effects through the Ang II type 2 (AT2) receptor as demonstrated by enhancement of MMP-1 production by an AT2 agonist and inhibition of MMP-1 production by an AT2 antagonist. Additionally, exogenous Ang II caused a significant enhancement in MMP-1 production by cytokine-stimulated monocytes. Furthermore, Ang II and the AT2 agonist increased PGE2 production, which in turn mediated the increase in MMP-1, as shown by the inhibition of MMP-1 by indomethacin or aspirin. In contrast, the AT2 antagonist inhibited the PGE2 induced by cytokines which account, in part, for the suppression of MMP-1 production. In a third area we have demonstrated that C reactive protein (CRP), a biomarker of inflammation and cardiovascular disease, enhances MMP-1 production by cytokine activated monocytes. Augmentation of MMP-1 by CRP occurs, in part, through the induction of an intermediate, monocyte chemotactic protein-1 (MCP-1). The role of MCP-1 in the enhancement by MMP-1 by CRP was demonstrated by suppression of MMP-1 by neutralizing antibodies against MCP-1 and the ability of exogenous MCP-1 to increase MMP-1 production by cytokine stimulated monocytes. In addition to CRP, oxidized LDL (ox-LDL) is another biomarker of atherosclerosis and potentially other diseases. When cytokine activated monocytes were exposed to CRP and ox-LDL there was a synergistic enhancement of MMP-1. The synergistic increase in MMP-1 by CRP and ox-LDL resulted from a differential regulation through MCP-1 and PGE2, respectively. Signaling events involved in proinflammatory pathways leading to MMP production by monocytes The CD40 receptor, a member of the TNFR superfamily, on monocytes through its interaction with CD40 ligand (CD154) on lymphoid cells leads to the secretion of inflammatory cytokines, chemokines, and MMPs. Our ongoing collaborative studies with investigators at the University of Louisville School of Medicine have demonstrated that TNFR-associated factor (TRAF) 6, a specific component of the CD40 receptor, is responsible for the induction of proinflammatory responses in monocytes. Since CD40 does not contain cytoplasmic sequences with catalytic activity, these findings demonstrate that TRAF6 acts as a critical CD40 adapter for downstream signaling components involved in proinflammatory pathways in monocytes and macrophages. Signaling pathways for the production of monocyte MMPs may differ based on the specific MMP and stimulus. Our study published in FY 05 demonstrates that lipopolysaccharide (LPS), a cell wall product of gram negative bacteria, induces MMP-9/gelatinase B through a specific pathway (phosphatidylinositol-3 kinase (PI-3K)/Akt/IKK alpha/NF-kappaB pathway). This pathway was defined through the use of specific inhibitors and by immunoprecipitation that demonstrated the correlation between the effect of these inhibitors on the phosphorylation of their specific targets and MMP-9 production. This is the first demonstration that Akt is involved in the signaling pathway leading to the production of monocyte MMP-9 and provides an additional approach in the regulation of this enzyme in human primary monocytes. Reactive oxygen species such as hydrogen peroxide (H2O2) have been associated with the initiation or aggravation of diverse pathological states. In a manuscript in press we report that addition of H2O2 to LPS activated monocytes, but not alone, caused a significant enhancement of the LPS-induced production of MMP-1, cyclooxygenase-2 (COX-2), and PGE2. The mechanism by which H2O2 increased these mediators was through the enhancement of IkappaB degradation, the inhibitor of the activation of the transcription factor NF-kappaB. The subsequent increase in NF-kappaB activation by H2O2 is responsible for the increase in MMP-1 production. The effect of H2O2 on IkB degradation and NF-kappaB activation was demonstrated through studies of co-immunprecipitation of IkB with p50 and ELISA assay of NF-kappaB p65 activity. The key role for NF-kappaB in this process was demonstrated by the ability of MG-132 or lactacystin (NF-kappaB inhibitors) to block the enhanced production of MMP-1, COX-2 and PGE2. In contrast, indomethacin, which inhibited PGE2 production, partially blocked the enhanced MMP-1 production. Moreover, while PGE2 restored MMP-1 production in indomethacin treated monocyte cultures it failed to significantly restore MMP-1 production in NF-kappaB inhibitor treated cultures. Thus in the presence of LPS and H2O2, NF-kappaB plays a dominate role in the regulation of production of MMP-1, COX-2 and PGE2 as well as in the PGE2 induction of MMP-1